Project Summary The rationale for the proposed research is based on the need for minimally invasive strategies to provide functional and aesthetic reconstruction in complex craniofacial defects caused by trauma and disease. The objective of this research is to develop an injectable, in situ forming hydrogel that utilizes a unique dual gelation mechanism to rapidly form gels at physiological temperature, chemically crosslink to maintain hydrogel stability, and biodegrade for full tissue regeneration. The proposed research will test the fundamental hypothesis that the injectable hydrogel will provide a supportive and mineralized substrate for mesenchymal stem cell (MSC) proliferation, enable osteogenic differentiation in vitro, and promote bone regeneration in vivo. The proposed research will be accomplished through three specific aims: 1) To develop and characterize injectable, in situ forming, biodegradable and space-filling hydrogels as an acellular tissue engineering therapeutic; 2) To assess the effectiveness of the injectable, dual-gelling hydrogels to promote the proliferation and osteogenic differentiation of encapsulated MSCs in vitro and bone regeneration in vivo; and 3) To evaluate the effects of cell seeding density and predifferentiation stage on the regenerative capacity of injectable, dual-gelling hydrogels in vivo. The novel macromers with crosslinkable and degradable functional groups will be synthesized and characterized using 1HNMR spectroscopy, differential scanning calorimetry, and rheometry. Cytocompatibility, encapsulated MSC viability and in vitro osteogenic differentiation will be measured via real-time PCR for osteogenic marker gene expression and essential biochemical assays for cell proliferation, mineralized extracellular matrix production and alkaline phosphatase activity. In order to evaluate the capacity of the hydrogels to promote bone regeneration, histological and histomorphometric scoring, microcomputed tomography analysis and mechanical testing will be used to quantify the tissue response and characterize functional bone formation. At the completion of these studies, the expected outcomes are successful fabrication of an in situ forming, injectable hydrogel capable of biodegradation, biomineralization, and stem cell delivery and the extensive evaluation of the system's efficacy for bone regeneration. The proposed work will not only improve the understanding and testing of biomaterials-based therapies for minimally invasive tissue regeneration as viable clinical alternatives, but provide new insights in the rational design of thermosensitive materials and hydrogel biomineralization. Moreover, the proposed system provides a novel platform for composite tissue regeneration and controlled delivery for application in a variety of different tissues.